JPH073420B2 - Analytical element - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an analytical element, particularly an analytical element for analyzing a specific component in a fluid, and more specifically, a specific component in a biological fluid sample in a reduced form. The present invention relates to a dry analytical element for analyzing via an enzyme.

Background of the Invention [0002] Conventionally, many methods for analyzing a specific component in a fluid sample have been developed. These methods are roughly classified into a reaction system in which a reaction is performed in a solution and a reaction in a solid phase carrier. It can be divided into two types. The analytical reaction in a solution system (hereinafter referred to as wet chemistry) is widely known from a method called a manual method that does not use any machine to an automatic analytical instrument. Especially in the field of clinical chemistry, the progress has been remarkable, and in recent years, various automatic quantitative analyzers for clinical examination have been introduced into clinical laboratories of hospitals and the like.

However, the above-mentioned method has various drawbacks since the reaction is basically carried out in the form of an aqueous solution. That is, a large amount of water, particularly purified pure water or distilled water, is required in the analysis process, resulting in an increase in energy consumption. In addition, various automatic analyzers are extremely expensive in themselves, require a great deal of skill in their operation, require a huge amount of time and labor, and their waste liquids necessarily cause environmental pollution. It has drawbacks.

On the other hand, the analytical reaction in the solid phase system (hereinafter referred to as dry
It is called chemistry. ) Is also widely used, these are carried out by impregnating a filter paper or the like with a reagent.

The filter paper is made by impregnating a water-absorbent fibrous carrier such as a filter paper with a reagent solution and drying it as described in US Pat. No. 3,050,373 or US Pat. No. 3,061,523. These are generally referred to as analytical test strips or simply test strips.A fluid sample is dropped on the test strip, or the test strip is immersed in the fluid sample, and the color change or concentration change of the test strip is visually judged, or It is measured by a reflection densitometer to determine the concentration level of the specific component in the fluid sample.

These test pieces are useful because they are easy to handle and the results can be obtained immediately, but due to their constitution, they remain in the area of semi-quantitative or qualitative analysis.

On the other hand, there is known a multi-layer analytical element that uses dry chemistry, which has a simple operability as compared with the conventional analytical method as described above, and has high quantification. For example, Japanese Patent Publication Sho 53-2
1677, JP-A-55-164356, JP-A-57-125847, Japanese Patent Application No. 56
-65446 and 56-189784, etc. describe multilayer analysis elements.

According to the elements described in these, all the reagents used in the analytical reaction are contained in one analytical element, and serum or whole blood is dropped onto the above-mentioned element in a constant volume and at a constant temperature for a predetermined time. After keeping the temperature, it is possible to measure the reflection density from the support side and determine the substance density from the reflection density.

The above-mentioned method has a remarkably high analytical precision as compared with the conventional test paper type, and has a performance equal to or higher than that of wet chemistry without preparing a reagent in advance.

In particular, a method of measuring a component in a fluid sample by increasing or decreasing reduced coenzyme, that is, reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate, has a wide application range and is a useful method. Known to be.

These reduced coenzymes are applied in wet chemistry, and after a specific component in a sample is mediated by a certain desired reaction pathway, it leads to a reduction reaction of the reduced coenzyme described above. A method of detecting by measuring the ultraviolet region by the initial velocity method, or by transmitting the change of reduced coenzyme to the pigment-forming precursor through the electron transfer agent, a pigment is formed, and the concentration of this pigment is determined. A method of quantifying by a colorimetric method is known.

The former method is generally used in wet chemistry, but there are some fatal problems in introducing it into the above-mentioned dry chemistry analysis element. That is, it is known that the change of the reduced coenzyme to be measured requires the absorption of ultraviolet light at 340 nm to be measured, and the molecular extinction coefficient thereof is extremely small. Therefore, it is necessary to measure minute changes in the absorbance in the ultraviolet region, and because of the structure of the analytical element, reflection photometry must be performed, so more sophisticated measuring equipment is required, and extremely expensive measuring equipment is used. There must be. Furthermore, in order to measure the ultraviolet region, it is difficult to use all materials that do not have absorption around 340 nm.

The latter method is not only far more advantageous than the former method because it forms a dye through an electron transfer agent and can be quantified by a colorimetric method in the visible part, and also has an initial rate method and a reaction end point. It is extremely advantageous because both methods can be used.

Despite this advantage, the latter method has not been used extensively in wet chemistry. The above-mentioned electron transfer agent and dye-forming precursor, when both of them coexist in a solution system, their stability is extremely lowered,
Not only does it have the serious drawback of inducing the formation of undesired dyes, it also suffers from poor precision when mixing the two and dyes derived from dye-forming precursors are often insoluble in water, Precipitation occurs in an aqueous solution, which causes problems such as deterioration of accuracy and deterioration of reproducibility due to the precipitation.

Therefore, when the latter method is simply applied to the multilayer analytical element, not only the usefulness inherent in the multilayer analytical element itself cannot be exhibited, but also the stability of the reagent and the measurement accuracy can be sufficiently satisfied. Obviously not.

For this reason, an analysis element applying the latter method while maintaining the usefulness of the multilayer analysis element is proposed in Japanese Patent Laid-Open No. 59-44658.

However, it cannot be said that the analytical element disclosed in the above publication has sufficient storage stability and color uniformity in the color development region.

[Object of the Invention] Accordingly, an object of the present invention is to provide an analytical element which is excellent in reagent stability and measurement accuracy and which can easily analyze a specific component in a biological fluid sample via a reduced coenzyme. To do.

[Structure of the Invention] That is, the above object of the present invention comprises a first reagent layer, a second reagent layer and a porous spreading layer on a light-permeable and liquid-impermeable support, and an electron transfer agent, at least A dye forming precursor, at least one oxidized coenzyme, at least one buffer and at least one reagent capable of converting the oxidized coenzyme to a reduced coenzyme through a specific component in a liquid sample. In an analytical element for analyzing a specific component in a liquid sample containing the buffer and the dye-forming precursor in different layers, the layer containing the buffer has the following general formulas (I) and (II It is achieved by using at least one binder selected from

General formula (I) [In the formula, R ₁ represents a methyl group or an ethyl group, x ₁ represents 20 to 95 mole percent, and y ₁ represents 5 to 80 mole percent. ] General formula (II) [In the formula, R ₂ represents a hydrogen atom or a methyl group, X represents a phenyl group, a cyano group or a —COOR ₃ group, and R ₃ represents a methyl group, an ethyl group or a butyl group. x ₂ represents 50 to 95 mole percent and y ₂ represents 50 to 5 mole percent. [Specific Configuration of the Invention] The electron transfer agent according to the present invention is produced by the reaction of at least one of the reagents according to the present invention and an oxidized coenzyme with the presence of a specific component in a biological fluid sample. The electron transfer agent, which has been reduced in the presence of the reduced coenzyme, which is further reduced, reduces the dye-forming precursor and forms a dye having absorption in the visible region.

Specific components in the fluid sample that can be measured in the present invention include, for example, lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), amylase (AM
Y), creatine phosphokinase (CPK), triglyceride (TG) and the like.

The reagent according to the present invention includes an enzyme, a pigment-forming precursor, a buffer, a coenzyme, and optionally a substrate.

These reagents are appropriately selected depending on the specific component in the biological fluid sample to be measured, and for example, lactic acid when measuring LDH, aspartic acid when GOT, α-ketoglutarate and glutamate dehydrogenase (GlDH). , GPT, alanine, α-ketoglutarate and glutamate dehydrogenase (GlDH), AMY, maltopentose, orthophosphate, β-phosphoglucomutase (β-PG
M), glucose oxidase (GOD) and maltose phosphorylase (MP), creatine in the case of CPK, adenosine triphosphate (ATP), hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6- PD
H), TG for lipoprotein reversase (LPL), glycerokinase (GK), glycerophosphate dehydrogenase (GPD)
H) and adenosine triphosphate (ATP).

The oxidized coenzyme according to the present invention refers to oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) and oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) and the like. The reduced coenzyme is a reduced form of the oxidized coenzyme. The reduced form of NAD ⁺ is NADH and the reduced form of NADP ⁺ is NADPH.

Below, a reaction formula in which a reduced coenzyme is produced by the reaction of at least one of the reagents according to the present invention with an oxidative coenzyme through a specific component in the biological fluid sample according to the present invention is shown. Show.

LDH GOT GPT AMY CPK TG Examples of the electron transfer agent used in the present invention include N-methylphenazine methosulfates (for example, N-methylphenazine methosulfate, 1-methoxy-5-methylphenazine methosulfate, etc.), Meldola blue, methylene blue, diaphorase, etc. Can be used, and preferable electron transfer agents include N-methylphenazine methosulfates and diaphorase.

On the other hand, tetrazolium salts are usually used as the dye-forming precursor according to the present invention. Most of the tetrazolium salts used in the present invention become poorly soluble or insoluble in water after dye formation, which is usually wet.
Although it is difficult to use by the chemistry method, it can be preferably used because the dye formed is resistant to diffusion, prevents unwanted ringing, and improves the quantitativeness of measurement.

Examples of the tetrazolium salt useful in the present invention include, for example, 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis [2- (p-nitrophenyl) -5.
-Phenyltetrazolium chloride, 3,3 '-(3,3'-
Dimethoxy-4,4'-biphenylene) -bis [2,5-diphenyltetrazolium chloride], 3- (4'5'-dimethyl-2-thiazolyl) -2,4-diphenyltetrazolium bromide, 3- (p-iodo) Phenyl) -2-
(P-Nitrophenyl) -5-phenyl-tetrazolium chloride, 2,2 ', 5,5'-tetra- (p-nitrophenyl) -3,3'-(3-dimethoxy-4-diphenylene) -ditetrazolium Chloride, 2,3,5-triphenyltetrazolium chloride, 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis- [2,5-bis (p-
Nitrophenyl) tetrazolium chloride and 3,3 ′
Examples thereof include-(4,4'-biphenylene) -bis [2,5-diphenyltetrazolium chloride].

Among the above-mentioned tetrazolium salts, those preferably used are 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis [2- (p-nitrophenyl) -5-phenyltetrazolium chloride. ] And 3,3 '-(4,4'-
Biphenylene) -bis [2,5-diphenyltetrazolium chloride] can be mentioned, and it is preferably contained in the first reagent layer according to the present invention.

The buffer used in the present invention is appropriately selected depending on the optimum pH in the above reaction. For example, Tris buffer (known as a combination of trishydroxymethylaminomethane and trishydroxyaminomethane hydrochloride), known as Good's buffer, carbonate buffer, etc. can be preferably used.

The buffering agent is preferably contained in a layer different from the above-mentioned dye-forming precursor. It goes without saying that these are laminated in a state where they are not mixed at the time of production and application of the sample. Therefore, it is preferable that the above-mentioned buffer is dispersed in the binder.

As the binder according to the present invention, a copolymer represented by the following general formula can be used.

General formula (I) [In the formula, R ₁ represents a methyl group or an ethyl group, x ₁ represents 20 to 90 mole percent, and y ₁ represents 5 to 80 mole percent. ] General formula (II) [In the formula, R ₂ represents a hydrogen atom or a methyl group, and X represents a substituted or unsubstituted phenyl group, a cyano group or a —COOR ₃ group. R ₃ represents a methyl group, an ethyl group or a butyl group, x ₂
Represents 50 to 95 mole percent and y ₂ represents 50 to 5 mole percent.

Typical examples of the copolymer according to the present invention are shown below, but the present invention is not limited thereto.

Exemplified polymer (1) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 95: 5) (2) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 80:20) (3) N-vinylpyrrolidone -Vinyl acetate copolymer (molar ratio 70:30) (4) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 60:40) (5) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 50 : 50) (6) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 40:60) (7) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 30:70) (8) N-vinylpyrrolidone -Vinyl acetate copolymer (molar ratio 20:80) (9) N-vinylpyrrolidone-methyl acrylate copolymer (molar ratio 95: 5) (10) N-vinylpyrrolidone-methyl acrylate copolymer (mol Ratio 90:10) (11) N-vinylpyrrolidone-methyl acrylate Combined (molar ratio 75:25) (12) N-vinylpyrrolidone-methyl methacrylate copolymer (molar ratio 90:10) (13) N-vinylpyrrolidone-acrylic acid-n-butyl copolymer (molar ratio 90:10) (14) N-vinylpyrrolidone-acrylonitrile copolymer (molar ratio 85:15) (15) N-vinylpyrrolidone-styrene copolymer (molar ratio 90:10) (16) N-vinylpyrrolidone- Styrene copolymer (molar ratio 75:25) (17) N-vinylpyrrolidone-styrene copolymer (molar ratio 50:50) The polymer according to the present invention can be easily obtained by a known polymerization method. it can.

By forming the layer of the copolymer of the present invention in combination with the buffer, for example, not only storage stability is improved, but also the uniformity of coloration in the color development region is improved, resulting in simultaneous reproducibility. Significant improvements can be brought about.

The reagent and oxidized coenzyme according to the present invention contained in the analytical element of the present invention may be contained in any of at least one reagent layer and at least one spreading layer.
Further, the electron transfer agent and the dye-forming precursor may be contained in any one of the at least one reagent layer and the at least one developing layer, respectively. However, if the electron transfer agent is other than diaphorase, the electron transfer agent and the chromogenic precursor will not react with each other until the fluid sample is applied so that they do not form undesired dyes. Must be contained in. Therefore, it is preferable that the electron transfer agent other than diaphorase and the dye-forming precursor are contained in different layers of at least one reagent layer and at least one developing layer. Alternatively, even when contained in the same layer among at least one reagent layer and at least one development layer, these electron transfer agent and dye-forming precursor are contained as separate particles, respectively. It is preferable.

However, this is not the case when diaphorase is used as the electron transfer agent, and for example, diaphorase and the dye-forming precursor can be contained in the same layer.

An electron transfer agent and a dye-forming precursor are contained in different layers, and an electron transfer agent is contained in any one or more layers of at least one reagent layer and at least one developing layer, and no electron transfer agent is contained. In the analysis element containing the dye-forming precursor in any one of the layers, even if the electron transfer agent is contained in any one of the spreading layers and the dye-forming precursor is contained in the reagent layer. Although it may be contained in the opposite state, it is preferably the former state. In this case, it is more preferable that the layer containing neither the electron transfer agent nor the dye-forming precursor is interposed between the developing layer containing the electron transfer agent and the reagent layer containing the dye-forming precursor.

When an electron transfer agent other than diaphorase is contained in the spreading layer, it can be generally contained by dispersion or dissolution, but since the content of the electron transfer agent is extremely small, it spreads sufficiently uniformly when dispersion is used. Difficult to include in the layer. In addition, in the case of dissolution, there is a possibility of shifting to a multilayer in the manufacturing process, and as a result, there is a possibility of causing deterioration of performance by contact with a compound which is not preferable to coexist with an electron transfer agent. In this case, the fiber structure developing layer disclosed in JP-A-57-197466 can be advantageously used. That is, after adding the electron transfer agent solution to a coating solvent such as xylene, the fibers are added individually to impregnate the above electron transfer agents into the fibers, and then a filter-dried product is used to accurately measure a small amount of electron transfer agents. In addition, it can be contained while preventing the transition to a multilayer.

When the electron transfer agent and the dye-forming precursor are contained in different layers, a method for providing these containing layers is to dissolve the electron transfer agent or the dye-forming precursor in an aqueous binder solution, and more specifically, After dissolving in water, this solution may be added to a binder dissolved in water and dissolved to be applied.

When an electron transfer agent and a dye-forming precursor are contained as separate particles in the same layer of at least one reagent layer and at least one developing layer, these electron transfer agent and dye-forming precursor are included. The particle size of the substance is 0.
It is preferably 1 μ or more.

Further, even when the electron transfer agent and the dye-forming precursor are contained in different layers, the electron transfer agent and / or the dye-forming precursor can be in the form of particles.

As a method of providing a layer containing an electron transfer agent and a dye-forming precursor as particles, a finely pulverized electron transfer agent and a dye-forming precursor are suspended in an organic solvent and dissolved in the same organic solvent. The obtained suspension is added to the binder and uniformly dispersed, or the electron transfer agent and the dye-forming precursor are suspended in an organic solvent and finely ground. , A method of applying a uniformly dispersed solution added to a binder dissolved in the same organic solvent, or the electron transfer agent and the dye-forming precursor, after finely pulverized in an organic solvent containing a binder, Examples include a method of applying a uniform material.

When the spreading layer is composed of a fibrous porous material such as a filter paper, after adding the electron transfer agent and the dye-forming precursor to the organic solvent containing the binder, the porous material is added and uniformly dispersed. The method of applying is mentioned.

The amount of the electron transfer agent used in the present invention contained in the analytical element of the present invention varies depending on the amount of the specific component, but in the case of an electron transfer agent other than diaphorase, it is usually 1 mg / m ² to.
It is 1 g / m ² , preferably 10 to 500 mg / m ² .

Furthermore, when diaphorase is used as an electron transfer agent, it varies depending not only on the amount of the specific component but also on the origin of diaphorase and the method for measuring the activity value, usually 100 U.
/ m ² ~100,000U / m ^2, preferably it is contained ^{500U / m 2 ~50,000U / m 2} .

The amount of the dye-forming precursor of the present invention contained in the analytical element of the present invention is usually 10 mg / m ^{2 to} 10 g / m ² , preferably 50 mg / m ^{2 to 3} g / m ² . Furthermore, the amount of the oxidized coenzyme according to the present invention contained in the analytical element of the present invention is usually 10 mg / m ^{2 to} 50 g / m ² , and preferably 50 mg / m ^{2 to} 10 g / m ² .

The binder used for forming the layer containing the electron transfer agent and the layer containing the dye-forming precursor is not particularly limited, but a hydrophilic colloid material is preferably used. For example, gelatin and its derivatives, polyacrylamide and its copolymers, polymethacrylamide and its copolymers, polyhydroxy, ethyl, acrylate and its copolymers, polyvinyl alcohol, agarose, carboxy, methyl, cellulose sodium salt, hydroxy. Examples include hydrophilic cellulose derivatives such as ethyl and cellulose. Gelatin and its derivatives are preferably used for the above purposes. The method for forming the layer is not particularly limited.

In the analytical element of the present invention thus configured, the storage stability of the fabricated element, including the fog density of the analytical element, is dramatically improved, and it becomes possible to obtain a high discriminating ability.

The spreading layer according to the present invention includes (1) a fixed volume of a fluid sample,
It has a function of uniformly distributing a fixed volume per unit area to the reagent layer. Furthermore, the performance described in JP-B-53-21677, that is, (2) the function of removing a substance or factor that inhibits the analytical reaction in the fluid sample, and / or (3) spectrophotometric analysis It is preferable that it has a function of performing a background action of reflecting the measurement light transmitted through the support when performing. Therefore, the development layer according to the present invention is a layer having only the function of (1) above,
In addition to (1), it may be any of the layers having the functions of (2) and / or (3) together, or a plurality of functions including (1) may be appropriately separated and each function may be separated. It is also possible to use another layer for. Further (1),
Of the functions (2) and (3), a layer having two functions and a layer having the remaining one function can be used in combination. For example, a development layer of a non-fibrous porous medium called a brush polymer composed of titanium dioxide and cellulose diacetate described in JP-B-53-21677 mentioned above, JP-A-56-24576 and JP-A-57-125847. No. 57-197466 and the like. In particular, the above-mentioned fibrous structure spreading layer is particularly useful as a material capable of promptly transporting blood cells as well, and is also useful for deploying and transporting macromolecules such as enzymes, which is one of the objects of the present invention. . The thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, but is preferably about 100 to about 500 microns, more preferably about 150 to 350 microns. Also, the porosity is preferably about 20 to about 85%.

The reagent layer according to the present invention is such that when a specific substance in a fluid sample finally passes through an electron transfer agent by a selected reaction and the dye-forming precursor is changed into a dye, the dye-forming precursor is It is provided for the purpose of serving as a place for receiving a dye that has changed into a dye in the reagent layer or at least from a dye-forming precursor and detecting it as a change in the spectroscopic observation amount. It is possible to contain various reagents and various additional additives as long as they do not conflict with the above.

As other additional additives, various additives such as preservatives and surfactants can be added as desired.

In particular, the surfactant can be effectively used for controlling the permeation rate when the fluid sample is applied to the device of the present invention.

As the usable surfactant, ionic (anionic or cationic) and nonionic surfactants can be used, but the nonionic surfactant is effective.
Examples of nonionic surfactants include, for example, 2,5-di-
Examples thereof include polyalkylene glycol derivatives of alkyl-substituted phenols such as t-butylphenoxy polyethylene glycol, p-octylphenoxy polyethylene glycol, p-isononylphenoxy polyethylene glycol, and higher fatty acid polyalkylene glycol esters. These surfactants have the effect of controlling the permeation rate of the fluid sample into the reagent layer and, at the same time, suppressing the occurrence of the undesirable "chromatographic phenomenon".

The surfactant may be used in a widely selected amount, but may be used in an amount of 25% by weight to 0.005% by weight, preferably 15% by weight to 0.05% by weight, based on the weight of the coating solution.

The liquid-impermeable light-permeable support according to the analytical element of the present invention (hereinafter, abbreviated as the support according to the present invention) is liquid-impermeable and light-transmissive. Various polymeric materials, such as, but not limited to, cellulose acetate, polyethylene terephthalate, polycarbonate or polystyrene, are suitable for this purpose. Furthermore, not only the above-mentioned polymer materials but also inorganic materials such as glass can be used as well. The thickness of the support according to the present invention is arbitrary, but is preferably about 50 to about 250 microns.
Further, one side surface of the support according to the present invention on the observation side can be optionally processed according to its eye. Further, a light-transmitting undercoat layer may be optionally used on the side of the support on which the reagent layer is laminated to improve the adhesion between the reagent layer and the support.

The analysis element of the present invention may be used, for example, as described in U.S. Pat.
992,158 reflective layer, undercoat layer, U.S. Pat.No. 4,042,3
Radiation blocking layer as described in US Pat. No. 4,066,40
No. 3 barrier layer, U.S. Pat.No. 4,166,093 migration prevention layer, JP-A-55-90859 described scavenger layer, U.S. Pat. The configuration can be arbitrary according to the purpose of the invention.

Various layers of these analytical elements are sequentially laminated on the support according to the present invention according to a desired constitution by appropriately selecting and using a slide hopper coating method, an extrusion coating method, a dip coating method, etc. which are conventionally known in the photographic industry. By doing so, a layer having an arbitrary thickness can be applied.

By using the analytical element of the present invention, the amount of the specific component in the fluid sample can be measured from the support side according to the present invention by reflection spectrophotometry according to the initial velocity method or the reaction end point method. The amount of the specific component can be determined by applying the measured value thus obtained to a calibration curve prepared in advance.

The amount of the fluid sample applied to the analytical element of the present invention can be arbitrarily determined, but is preferably about 5 μl to about 50 μl.
And more preferably 5 to 20 μl. Normal
It is preferred to apply 10 μl of fluid sample.

The analytical element of the present invention can be used for any analysis of whole blood, serum and plasma without any inconvenience. Furthermore, other body fluids such as urine, lymph fluid, and cerebrospinal fluid can be used without any inconvenience. If whole blood is used, the radiation blocking layer or other concealing layer described above can be provided, if desired, to prevent radiation for detection from being interfered with by the blood cells. The analytical reaction used in the analytical element of the present invention can be arbitrarily determined depending on its purpose, but is useful in the field of, for example, clinical chemistry, and particularly biological liquid samples, that is, blood (including serum, blood, etc.) Or used for analysis of components in urine.

As described in detail above, according to the analytical element of the present invention, until the fluid sample is applied, the electron transfer agent and the dye-forming precursor can be contained in the constituent layers in a state in which they cannot react with each other. As a result, the storage and stability of the reagent was improved, the fog density was reduced, the color development was excellent, and the occurrence of non-uniform density and chromatographic phenomenon were hardly seen. An ordinary spectrophotometer can be simply and quickly used for highly accurate dry quantitative analysis of components in a fluid sample, particularly a biological fluid sample, which is extremely practically advantageous.

Hereinafter, the present invention will be described in more detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1 A 180 μm undercoated transparent polyethylene terephthalate support was sequentially coated with layers having the following composition to give an analytical element.

First reagent layer Gelatin 18.9 g / m ² glutamate dehydrogenase 19,000 U / m ² diaphorase 1,900 U / m ² 3,3 ′-(3,3′-dimethoxy-4,4′-biphenylene)
-Bis [2,5-diphenyltetrazolium chloride] 0.9
4 g / m ² 1,2-bis (vinylsulfonyl) ethane 0.1 g / m ² Triton X-100 (Rohm & Hass) 0.1 g / m ² dry reagent thickness 23 μm First reagent layer Second reagent layer trishydroxymethylaminomethane 3.87 g / m ² HCl tris (hydroxymethyl) aminomethane 0.83 g / m ² Triton (Triton) X-100 0.1g / m 2 binder dry film thickness consisting of (Table 1 refer) 1.35 g / m ² Second reagent layer of about 15 μm Development layer Powder filter paper C [Toyo Filter Paper (manufactured by) 300 mesh or more] 8.92 g / m
² Alanine 2.9 g / m ² Oxidized nicotinamide dinucleotide 5.3 g / m ² α-Ketoglutaric acid 0.2 g / m ² Styrene-glycidyl methacrylate copolymer (weight ratio 9: 1) 15 g / m ² Triton Development layer composed of X-100 5 g / m ² and having a dry film thickness of about 250 μm As the binder of the second reagent layer, those shown in Table 1 below were used.

Various types of the comparative analysis element and the analysis elements I to IV of the present invention can be used.
After 10 μl of GPT-active human serum was added dropwise, the mixture was incubated at 37 ° C., and the reflection density after 2 minutes and 4 minutes after the addition was measured with a reflection spectrophotometer through a 546 nm filter, and the difference in the reflection density, Of human serum tested by standard method
A calibration curve was prepared in advance from the GPT activity value.

Furthermore, normal (25 carmen units) and abnormal (98 carmen units) of human serum were dropped 15 times each, and the same operation was performed to obtain the difference in reflection density, which was fitted to the calibration curve prepared in advance. , The standard deviation and CV were calculated after obtaining the activity value. The results are shown in Table-2.

As is clear from Table 2 above, the analytical element of the present invention is
Not only did good simultaneous reproducibility be exhibited, but the color development in the colored area was uniform. On the other hand, the comparative analytical element was inferior in the simultaneous reproducibility and inferior in the uniformity in the coloring region to the analytical element of the present invention.

Example-2 The analytical elements (I) to (IV) and the comparative analytical element of the present invention were stored at 40 ° C. and 55% relative humidity for 7 days, and then, a normal value (25 carmen unit) and an abnormal value (98 carmen) were obtained. (Unit :) human serum was added dropwise, and the same operation was performed to calculate the difference in reflection density, which was then fitted to a calibration curve on the same day, converted into an activity value, and the fluctuation rate was calculated. The results are shown in Table-3.

As is clear from Table 3 above, it is clear that the analytical element of the present invention has better storage stability than the comparative analytical element.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Seiji Hidaka No. 1 Sakura-cho, Hino-shi, Tokyo Photo Konishi Roku Photo Industry Co., Ltd. (72) Inventor Seiichi Sukao No. 1 Sakura-cho, Hino-shi, Tokyo Photo Roku Konishi Kogyo Co., Ltd. (56) Reference JP-A-58-171668 (JP, A) JP-A-57-50660 (JP, A)

Claims (1)

[Claims] 1. A first reagent layer, a second reagent layer and a porous spreading layer on a light-permeable and liquid-impermeable support,
The oxidized coenzyme can be converted to a reduced coenzyme via an electron transfer agent, at least one pigment-forming precursor, at least one oxidized coenzyme, at least one buffer and a specific component in a liquid sample. In an analysis element for analyzing a specific component in a liquid sample containing at least one reagent and having the buffer and the dye-forming precursor arranged in different layers, the layer containing the buffer has the following general formula ( I)
And an analytical element for analyzing a specific component in a liquid sample, characterized by using at least one binder selected from (II). General formula (I) [In the formula, R ₁ represents a methyl group or an ethyl group, x ₁ represents 20 to 95 mole percent, and y ₁ represents 5 to 80 mole percent. ] General formula (II) [In the formula, R ₂ represents a hydrogen atom or a methyl group, X represents a phenyl group, a cyano group or a —COOR ₃ group, and R ₃ represents a methyl group, an ethyl group or a butyl group. x ₂ represents 50 to 95 mole percent and y ₂ represents 50 to 5 mole percent. ]